In the current funding period, using human and mouse monoclonal antibodies, we were the first to define and partially characterize three separate epitope clusters on gpl20 important for antibody neutralization of HIV-1. These epitopes, including one cluster which is conserved but conformation-dependent (discontinuous), are distinct from the well-known neutralization site in the V3 domain of gpl20. In order to further the effort of vaccine development and enhance the strategy of immunotherapy, we now propose to map the epitopes of five neutralizing monoclonal antibodies that recognize discontinuous elements on gpl20 using three parallel approaches. First, we will examine the reactivity of these monoclonal antibodies in serologic assays with gpl20 of envelope mutants created by site-directed mutagenesis. Second, we will generate antibody escape mutants in vitro followed by nucleotide sequencing of the envelope of escaped viruses. Third, we will obtain HIV-1 quasispecies (from the same patient whose B cells produce the human monoclonal antibodies) by biologic cloning and determine their susceptibility to antibody neutralization. Given the high degree of sequence homology seen among quasispecies, specific amino acid sequences consistently found in neutralization-sensitive clones and not found in neutralization-resistant clones may aid the identification of the antibody epitopes. Collectively, these approaches should yield important structural information on non-V3 neutralization sites on gpl20, the major target in the AIDS vaccine effort. We also propose to serotype 100 U.S. strains of HIV-1 using our well-characterized panel of neutralizing monoclonal antibodies and large collection of primary viral isolates. Although extremely laborious and seemingly limited in scope, this systematic effort should provide valuable and useful information on antigenic diversity of HIV-1 isolates in the United States. The successful identification of major antigenic types (serotypes) of HIV-1 will have important implications for vaccine development and immunotherapy.